阅读说明：本技术 一种利用微生物转化生产7α-羟基-脱氢表雄酮的方法 (Method for producing 7 α -hydroxy-dehydroepiandrosterone by microbial transformation ) 是由 申雁冰 王敏 骆健美 邓铭倩 王喜波 王璐 李梦涵 夏梦雷 于 2019-12-24 设计创作，主要内容包括：本发明属于微生物甾体转化技术领域,公开了一种7α-OH-DHEA的生物合成方法。涉及生产菌株为蓝色犁头霉(Absidia coerulea),具体为蓝色犁头霉CGMCC 14124,生产原料为脱氢表雄酮(DHEA)。利用该菌种的选择性羟基化能力对DHEA进行生物转化,可以有效合成目标产物7α-OH-DHEA。底物DHEA的投料浓度高达5.5g/L时,目标产物的生成率达到了45.5％。该方法实现了绿色环保的生产,为该甾体药物的工业化生产提供了基础。(The invention belongs to the technical field of microbial steroid conversion, and discloses a biosynthesis method of 7 α -OH-DHEA, which relates to a production strain of Absidia coerulea (Absidia coerulea), specifically Absidia coerulea CGMCC14124, wherein a production raw material is Dehydroepiandrosterone (DHEA). The DHEA is biologically converted by utilizing the selective hydroxylation capacity of the strain, a target product 7 α -OH-DHEA can be effectively synthesized, when the feeding concentration of a substrate DHEA is up to 5.5g/L, the generation rate of the target product reaches 45.5%, the method realizes green and environment-friendly production, and provides a foundation for the industrial production of steroid medicines.)

1. A method for producing 7 α -OH-DHEA by using microorganisms is characterized in that Absidia coerulea is taken as a production strain, and a product 7 α -OH-DHEA is obtained by converting a substrate DHEA after spore suspension preparation, seed culture, fermentation culture and conversion condition improvement, and the method comprises the following specific steps:

transferring the Absidia coerulea AL-172 seed solution into a fermentation medium with the inoculation amount of 10% -15%, fermenting and culturing at 25-30 ℃ for 5-8h at 200r/min, then using 0.3-1.0g/L DHEA for induction, adjusting the pH of the fermentation liquid to 5.6-6.0 when the pH of the culture is 3.8-4.2, simultaneously adding 2-5g/L DHEA into the fermentation medium, converting for 5-7h, adjusting the pH of the fermentation liquid to 5.4-6.0 again, supplementing residual substrates to make the total feeding concentration reach 3-8g/L, and continuing to convert. Sampling the fermentation liquor at regular time in the conversion process, and measuring the product generation rate.

2. The method for producing 7 α -OH-DHEA by using a microorganism as claimed in claim, wherein the specific strain is Absidia coerulea (Absidia coerulea) AL-172, with the accession number of CGMCC 14124.

3. The method of claim 1 wherein the seed medium component (g/L) is corn steep liquor 12, glucose 10.5, ammonium sulfate 5, yeast extract 2-3, water, pH 6.5.

Fermentation medium composition (g/L): 12 parts of corn steep liquor, 11 parts of glucose, 5 parts of ammonium sulfate and 2-3 parts of yeast extract; water, pH 6.5.

4. The method for producing 7 α -OH-DHEA by using the microorganism as claimed in claim 1, wherein the seed culture solution is prepared by inoculating 1mL of Absidia coerulea spore suspension into a seed culture medium (30/250mL triangular flask), and culturing at 26-30 ℃ and 180-210r/min until the pH reaches 3.6-4.0.

5. The method of claim 1, wherein the spore suspension is prepared by washing slant spores of the strain with sterile water, filtering with 6 layers of gauze, shaking, counting with a hemocytometer, and adjusting the concentration of the spore suspension to 3-10X 10⁷one/mL.

The technical field is as follows:

the invention relates to the technical field of microbial steroid conversion and industrial fungi, in particular to a method for preparing 7 α -OH-DHEA by converting DHEA through Absidia coerulea to generate 7 α -OH-DHEA.

Background art:

dehydroepiandrosterone (DHEA) is an important active substance in a natural organism and has an important regulation effect on vital metabolic activity, and a hydroxylation product 7 α -OH-DHEA is medically used for controlling weight and treating Alzheimer's disease, has wide application, is high-efficiency and low-toxicity, is an important steroid medicament, and has great market demand and great application value.

At present, dehydroepiandrosterone acetate is used as a raw material for chemically synthesizing 7 α -OH-DHEA in industry, two main methods are to obtain a target product through bromination, isomerization, esterification and hydrolysis, the process is complicated, acyloxy is introduced into the position 7 α by substituting the free radical of benzoyl peroxide tert-butyl ester, and the product is obtained through hydrolysis.

The microbial transformation has many advantages compared with chemical synthesis medicines, the microbial transformation can generate a plurality of substances with unique chemical structures, the chemical synthesis way is difficult to obtain, the microbial growth cycle is short, the microbial transformation can be easily regulated and controlled, and the industrial production can be realized, the existing strains for transforming DHEA7 α hydroxylation mainly comprise colletotrichum lini, mucor racemosus, gibberella and the like, but the problems of low feeding concentration, more byproducts and the like exist, and the industrial production requirements cannot be met.

The invention content is as follows:

in order to achieve the purpose, the invention provides a process for producing 7 α -OH-DHEA by high-efficiency biotransformation, in particular to a biotransformation process for carrying out DHEA7 α hydroxylation under the condition of high feeding concentration by using a strain of Laurella coerulea with excellent performance, wherein the purpose of the invention is realized by the following technical scheme:

the Absidia coerulea strain AL-172(Absidia coerulea) with excellent properties has the microbial preservation number of CGMCC14124, the preservation time of 2017, 5 and 11 months, the preservation unit is China general microbiological culture Collection center, and the method for converting DHEA into 7 α -OH-DHEA by applying the strain comprises the following steps:

transferring the Absidia coerulea AL-172 seed solution into a fermentation medium with the inoculation amount of 10% -15%, fermenting and culturing at 25-30 ℃ for 5-8h at 200r/min, then firstly using 0.3-1.0g/L DHEA to induce, when the culture pH is 3.8-4.2, adjusting the pH of the fermentation liquid to 5.6-6.0, simultaneously adding the DHEA with the concentration of 2-5g/L into the fermentation medium, converting for 5-7h, then adjusting the pH of the fermentation liquid to 5.4-6.0 again, supplementing residual substrates to make the total feeding concentration reach 3-8g/L, and sampling at regular time during the period, wherein the total conversion period is 48 h. Extracting the fermentation liquor by using an organic solvent, taking supernatant, and detecting the conversion condition by using high performance liquid chromatography.

The seed culture medium comprises the following components (g/L): 12 parts of corn steep liquor, 10.5 parts of glucose, 5 parts of ammonium sulfate and 2-3 parts of yeast extract; water, pH 6.5. The fermentation medium had the following composition (g/L): 12 parts of corn steep liquor, 11 parts of glucose, 5 parts of ammonium sulfate and 2-3 parts of yeast extract; deionized water, pH 6.5.

The culture conditions of the seed liquid are as follows: 1ml of spore suspension is inoculated in a seed culture medium and cultured under the conditions of 26-30 ℃ and the rotating speed of 170-220r/min until the pH value of a culture solution reaches 3.8-4.4.

The sporeThe suspension was prepared as follows: washing slant hypha with sterile water, pouring suspension containing large amount of spores into a triangular flask attached with 4 layers of sterilized gauze and filled with glass beads, and shaking sufficiently to obtain a concentrate with a concentration of 3-10 × 10⁷Spore suspension of one/ml.

Preferably, the substrate DHEA is converted to a final concentration of 5-6g/L.

Preferably, DHEA is added in an amount of 0.4-0.6g/L when the substrate DHEA is added for induction.

The method has the beneficial effects that the problem of low feeding concentration in the DHEA7 α hydroxylation reaction is solved, and when the feeding concentration of the substrate DHEA is up to 5.5g/L, the generation rate of the target product reaches 45.5%.

Description of the drawings:

FIG. 1 is a schematic diagram of Absidia coerulea CGMCC14124 for producing 7 α -hydroxy-dehydroepiandrosterone.

FIG. 2 shows NMR H spectrum of product 7 α -hydroxy-dehydroepiandrosterone.

FIG. 3 is a graph of the process for the production of 7 α -hydroxy-dehydroepiandrosterone at high feed concentration (5.5 g/L).

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.